A growing wealth of data indicates that targeted therapies can mobilize a patient's own immune system to destroy malignancies with fewer side effects than traditional chemotherapy. Since it is estimated that 41% of Americans—almost 1 in 2 people—born in 2011 will develop cancer in their lifetime,8 the generation of more effective cancer immunotherapies is a high priority. These therapies include monoclonal antibodies (mAbs) that direct innate immune cells to tumor-associated antigens (TAA) as well as cancer “vaccines” that take many forms (including injections of tumor proteins with adjuvants or ex vivo primed dendritic cells) and are designed with the intention of inducing long-lasting anti-tumor T-cells.1 The objective of the present inventors' research is to develop novel compounds capable of stimulating both innate and adaptive immune responses against tumors, thereby combining the immunologic strengths of both mAbs and cancer vaccines in a small molecule format.
To date, mAbs have demonstrated the greatest clinical efficacy of all cancer immunotherapeutics, with nine unconjugated mAbs against TAA approved by the FDA over the past 15 years.9 To capitalize on this therapeutic success but reduce the disadvantages of mAbs (expensive to produce, can provoke allergic reactions, lack oral bioavailability), the present inventors have developed small-molecule antibody recruiting molecules (ARMs)2. ARMs take advantage of the high prevalence of preexisting antibodies against dinitrophenol (DNP) in human serum, possibly caused by pesticide exposure. These molecules redirect anti-DNP antibodies to prostate cancer and other cancer cells expressing prostate specific membrane antigen (PSMA). PSMA is a membrane protein expressed at high abundance in human prostate carcinoma, but present at low levels in normal prostate and non-prostatic tissues.10 We have shown that ARMs are able to mediate prostate cancer destruction both in vitro2 and in vivo11 through both antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) by innate effector cells.
While the traditional view holds that mAbs act primarily through the innate immune system, burgeoning evidence highlights their link to adaptive immunity.12 Antibody-opsonized tumors or soluble TAA immune complexes induce phagocytosis or endocytosis through Fcγ receptors (FcγRs) on dendritic cells (DCs).3, 4 This mode of internalization leads to increased presentation of TAA peptides on MHC I and II, increased costimulatory molecule expression, and increased generation of antigen-specific CD4+ and CD8+ T-cells as compared to unopsonized antigen.13-16 In mice, anti-CD20 and anti-Her2/neu mAbs were shown to induce adaptive anti-tumor immunity,17, 18 while in humans, clinical response following anti-Her2/neu therapy correlates with greater increases in endogenous anti-Her2/neu antibodies and anti-tumor CD4+ T-cells.19, 20 
Unfortunately, across all monoclonal antibodies used for tumor therapy, only about 30% of treated patients experience increased progression-free or overall survival.20, 21 The tumor microenvironment can be highly immunosuppressive, characterized by release of anti-inflammatory paracrine factors, consumption of metabolites necessary for Teff proliferation, and recruitment of tolerogenic cells such as regulatory T-cells (Tregs).5 Many tumors are also poorly immunogenic in that, despite their malignant transformation, they consist of mostly self-proteins to which the immune system is tolerant. Immunogenic epitopes arising from mutated gene products, expression of oncofetal proteins, or aberrant posttranslational modifications may represent only a small fraction of a tumor's total protein. Clearly, additional immunostimulation beyond unmodified anti-tumor mAbs is required to mount a successful adaptive immune response in the majority of patients.
Advances in the cancer vaccine field have underlined the importance of adjuvants in generating immunity Toll-like receptor (TLR) agonists are particularly powerful adjuvants, since the natural ligands of these receptors are highly conserved pathogen-associated molecular patterns that are recognized as primal danger signals by the immune system.22 pursuant to the present invention, the inventors propose to attach the synthetic small-molecule TLR7/8 agonist imiquimod and related TLR7/8 agonists to the ARM scaffold. Located in endosomes, TLRs 7/8 mediate production of proinflammatory cytokines and induce maturation of antigen presenting cells (APCs).6, 7 TLR7/8 agonists can heighten innate responses, especially through plasmacytoid dendritic cells, as well as potentiate adaptive Th1 polarization and cytotoxic T-cell (CTL) generation.23 Imiquimod is already FDA-approved for topical use against skin malignancies and has also been tested systemically as an experimental vaccine adjuvant.24, 25 It is the present inventors' view that the conjugation of imiquimod to an ARM will better stimulate APC activation and generate anti-tumor T-cells while also improving the ARM's ability to mediate tumor lysis by innate immune cells.
The direct conjugation of imiquimod and other TLR7/8 agonists to the ARM offers crucial advantages over co-administration of unlinked molecules. Several mouse studies have shown that antigen-specific CD4+ and CD8+ T-cell generation dramatically improved when injected antigen was directly linked to TLR7/8 agonists.26-28 Systemically administered TLR7/8 agonists in human trials have provoked significant adverse effects including fever, fatigue, nausea, chills, and myalgia without demonstrating much efficacy.29, 29, 30 Physical conjugation to the larger ARM should (1) increase the therapeutic index of the TLR7/8 agonist's (e.g. imiquimod's) therapeutic index by preventing its interaction with endosomal TLR7/8 until phagocytosis of an ARM-opsonized target cell has occurred and (2) localize the immunostimulant to the tumor site. Beyond this superior pharmacokinetic profile, there is some evidence that control of antigen presentation is phagosome-autonomous, meaning that TLR agonists must be located in the same endosome as phagocytosed TAA to best enhance TAA peptide presentation on MHC I or II.27, 31, 32 Extensive preclinical and clinical data thus support the conjugation of TLR7/8 agonists to antibody recruiting molecules for greater induction of anti-tumor immunity.
The Present Invention
The present invention relates to compounds which are designated TLR-ARMs. TLR-ARMs are multifunctional small molecules designed to stimulate both innate and adaptive anti-tumor immune responses. The inventors have previously developed bifunctional antibody recruiting molecules (ARMs) able to redirect endogenous antibodies to prostate cancer cells expressing prostate specific membrane antigen (PSMA). The present invention enhances the immunostimulatory properties of ARMs by attaching TLR agonists (potent pro-inflammatory molecules) to the ARM scaffold. The binding moieties from the parent compound target the immune response to cancer cells, in particular, prostate cancer cells, while the additional TLR agonistic motif activates local antigen presenting cells for induction of immunologic memory against the tumor. The result is an effect which provides synergistic anticancer activity which is substantially greater than the anticancer activity of individual functional molecules which are not linked through a multifunctional connector [MULLTICON] as in the present invention.
When prostate cancer is diagnosed prior to metastasis, the patient has a greater then 99% chance of survival. The most successful means for treating prostate cancer at this stage is a radical prostatectomy. Unfortunately, this surgery carries with it the risk of severing nerves and blood vessels associated with sexual organs and the bladder, and can potentially result in impotency and/or incontinency. Radiation therapy is yet another commonly used procedure that carries the risk of impotency. Half the patients who undergo radiation therapy for prostate cancer become impotent within 2 years of treatment. In addition to the adverse affects associated with these procedures, they are significantly less effective in patients whose cancer has already delocalized or metastasized on diagnosis. In these cases, patients generally undergo even more invasive procedures such as hormonal therapy or chemotherapy. Unfortunately, most patients eventually stop responding to hormonal therapy and the most successful chemotherapeutic, Taxotere, only prolongs the life of advanced prostate cancer patients by 2.5 months on average.
As discussed above, monoclonal antibody (mAb)-based immunotherapy has proven clinically beneficial for cancer patients while allowing them to maintain a good quality of life. These antibodies can either regulate proliferation of cancer cells through the manipulation of signal transduction, or promote cytotoxicity. Two examples of FDA-approved mAb-based anticancer drugs are Herceptin and Rituxan (Rituximab), which are currently being used for the treatment of breast cancer and non-Hodgkin's lymphoma, respectively. While there are no mAb-based therapeutics currently available for prostate cancer patients, advanced clinical studies on mAb-based immunotherapy has shown promise for the treatment of prostate cancer including advanced prostate cancer. Despite the major advantages of mAb-based immunotherapy, there are significant pitfalls which may limit its potential. In general, mAb-based therapeutics are highly costly ($70,000 for full course of treatment of Herceptin), lack oral bioavailability, and can lead to severe and often fatal side-effects. For example, Herceptin is associated with heart problems and cannot be administered to approximately 10% of cancer patients because of heart-related complications. Rituxan can cause several side-effects which include renal failure, infections and immune and pulmonary toxicity.
Now, no longer in its infancy, the concept of using small molecules to template the human immune response has shown realistic potential. Recent reports have surfaced in which small molecules have been used to direct antibodies to cancerous cells such as breast carcinoma cells, melanoma cells, and nasopharyngeal epidermal carcinoma cells. Animal studies have demonstrated that these molecules can promote tumor rejection and antitumor immunity in mice. Because this process allows for the direction of endogenous antibodies selectively to the cell of interest, it has the potential to harness the power of mAb-based therapeutics while limiting the costs and side effects associated with administering exogenous antibodies. By developing similar methods which recruit anti-DNP antibodies to prostate cancer cells, the proposed research will help broaden this field while creating a new therapy for all forms of prostate cancer, as well as numerous other cancers.